Biochemical modifications of gliadins induced by microbial transglutaminase on wheat flour

Background

Celiac disease (CD) is an immune-mediated disorder caused by the ingestion of wheat gluten. A lifelong, gluten-free diet is required to normalize the intestinal mucosa. We previously found that transamidation by microbial transglutaminase (mTGase) suppressed the gliadin-specific immune response in intestinal T-cell lines from CD patients and in models of gluten sensitivity.

Methods

SDS-PAGE, Western blot, ELISA, tissue transglutaminase (tTGase) assay and nano-HPLC–ESI-MS/MS experiments were used to analyze prolamins isolated from treated wheat flour.

Results

Gliadin and glutenin yields decreased to 7.6 ± 0.5% and 7.5 ± 0.3%, respectively, after a two-step transamidation reaction that produced a water-soluble protein fraction (spf). SDS-PAGE, Western blot and ELISA analyses confirmed the loss of immune cross-reactivity with anti-native gliadin antibodies in residual transamidated gliadins (K-gliadins) and spf as well as the occurrence of neo-epitopes. Nano-HPLC–ESI-MS/MS experiments identified some native and transamidated forms of celiacogenic peptides including p31–49 and confirmed that mTGase had similar stereo-specificity of tTGase. Those peptides resulted to be 100% and 57% modified in spf and K-gliadins, respectively. In particular, following transamidation p31–49 lost its ability to increase tTGase activity in Caco-2 cells. Finally, bread manufactured with transamidated flour had only minor changes in baking characteristics.

Conclusions

The two-step transamidation reaction modified the analyzed gliadin peptides, which are known to trigger CD, without influencing main technological properties.

General significance

Our data shed further light on a detoxification strategy alternative to the gluten free diet and may have important implications for the management of CD patients.     

2-氧代-4-苯基丁酸乙酯还原酶产生菌筛选及产酶条件

研究了利用生物催化不对称还原的方法制备(R)-2-羟基-4-苯基丁酸乙酯[(R)-HPBE]。以2-氧代-4-苯基丁酸乙酯(OPBE)为底物,通过对实验室保藏菌株进行筛选,得到一株产物立体选择性较高的菌株G2ndida krusei SW2026,并对其发酵产酶条件进行研究。其最适的发酵培养基组成为4.5%葡萄糖,3%蛋白胨,1.5%牛肉膏,0.05%Mn~(2+);适宜的产酶发酵条件为初始pH 6.0,温度28℃,摇床转速180 r/min,发酵周期48 h。将此条件下发酵培养的菌体用于OPBE的不对称还原反应,产物(R)-HPBE的对映体过量值(e.e.)可达97.33%,产率最高达到72.54%。     

Stereoselective formation of a 2′(3′)-aminoacyl ester of a nucleotide

Summary Reaction ofDl-serine and adenosine-5-phosphorimidazolide in the presence of adenosine-5-(O-methylphosphate) and imidazole resulted in the stereoselective synthesis of the aminoacyl nucleotide ester 2(3)-O-seryl-adenosine-5-(O-methylphosphate). The enantiomeric excess ofd-serine incorporated into 2(3)-O-seryl-adenosine-5-(O-methylphosphate) was about 9%. Adenylyl-(5N)-serine and an unknown product also incorporated an excess ofd-serine; however, serylserine showed an excess ofl-serine. The relationship of these results to the origin of the biological pairing ofl-amino acids and nucleotides containingd-ribose is discussed.     

阿托伐他汀对冠心病患者脂蛋白(a)及CETP水平的影响

目的:分析阿托伐他汀对冠心病患者脂蛋白(a)、血清胆固醇酯转运蛋白(CETP)水平的影响及临床疗效。方法:将112例冠心病患者随机分为对照组与观察组,每组56例。对照组患者采用辛伐他汀治疗,观察组患者采用阿托伐他汀治疗。观察并比较两组患者治疗前后血清LP(a),CETP,超敏C-反应蛋白(hs-CRP)及脑钠肽(BNP)水平,冠状动脉血流储备、舒张期峰流速及收缩期峰流速变化,左心室后壁厚度(LVPWT)、左心室收缩末期内径(LVESD)及左心室舒张末期内径(LVEDD)情况,以及临床疗效。结果:治疗后,观察组LP(a),CETP,hs-CRP及BNP水平均低于对照组,差异有统计学意义(P0.05);观察组冠状动脉血流储备、舒张期峰流速、收缩期峰流速均高于对照组,差异有统计学意义(P0.05);观察组LVPWT、LVESD、LVEDD均低于对照组,差异有统计学意义(P0.05);观察组总有效率高于对照组,差异有统计学意义(P0.05);两组安全性比较,差异无统计学意义(P0.05)。结论:阿托伐他汀对冠心病患者的临床疗效比较明确,可下调LP(a)及血清CETP表达。     

Lithium Selectively Inhibits Muscarinic Receptor-Stimulated Inositol Tetrakisphosphate Accumulation in Mouse Cerebral Cortex Slices

The in vitro effects of Li on agonist- and depolarization-stimulated accumulation of inositol phosphates were determined in mouse cerebral cortex slices. Of the agents examined, only the cholinergic agonist carbachol produced a significant accumulation of inositol tetrakisphosphate (InsP4) in the absence of Li. Lithium at 5 mM enhanced the accumulation of inositol monophosphate (InsP1) and inositol bisphosphate (InsP2) due to all the stimuli used and potentiated inositol trisphosphate (InsP3) accumulation due to histamine and noradrenaline, although at lower Li concentrations, carbachol-stimulated InsP3 accumulation was reduced. Li also enhanced InsP4 accumulation in the presence of noradrenaline, histamine, and elevated KCl level but, in marked contrast, reduced carbachol-stimulated InsP4 accumulation with an IC50 of 100 microM. There was a significant time delay between the initiation of carbachol stimulation and the beginning of the InsP4 inhibition due to Li. The phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate did not mimic the effects of Li. The results suggest that muscarinic receptor-mediated InsP4 production might be one of the targets for the therapeutic action of Li.     

改良法建立妊娠期高血压疾病大鼠模型

目的寻找理想的妊娠期高血压疾病动物模型。方法60只雌性Wistar大鼠分为六组,分别为正常妊娠组（A组）、左旋亚硝酸精氨酸甲酯（L-NAME）腹腔注射组（B组）、生理盐水注射组（C组）、假手术组（D组）、双侧子宫动脉结扎术组（E组）以及L-NAME腹腔注射联合腹主动脉缩窄术组（F组）,对大鼠血压、尿蛋白值以及胎鼠、胎盘、胎鼠头的重量进行监测并观察肾脏、胎盘的病理改变。结果F组孕鼠于孕13d即出现了蛋白尿、血压升高,较B组及E组出现时间早（P〈0.05）,其胎鼠体重、胎头重量下降较各组更明显（P〈0.05）,肾小球小动脉管壁增厚、管腔狭窄;胎盘出现血管间膜增厚、纤维蛋白沉积等妊娠高血压疾病的典型病理改变。结论L-NAME腹腔注射联合腹主动脉缩窄术是更为理想的建立妊娠高血压疾病动物模型的方法。     

Neurospora crassa FKBP22 is a novel ER chaperone and functionally cooperates with BiP

FK506 binding proteins (FKBPs) belong to the family of peptidyl prolyl cis-trans isomerases (PPIases) catalyzing the cis/trans isomerisation of Xaa-Pro bonds in oligopeptides and proteins. FKBPs are involved in folding, assembly and trafficking of proteins. However, only limited knowledge is available about the roles of FKBPs in the endoplasmic reticulum (ER) and their interaction with other proteins. Here we show the ER located Neurospora crassa FKBP22 to be a dimeric protein with PPIase and a novel chaperone activity. While the homodimerization of FKBP22 is mediated by its carboxy-terminal domain, the amino-terminal domain is a functional FKBP domain. The chaperone activity is mediated by the FKBP domain but is exhibited only by the full-length protein. We further demonstrate a direct interaction between FKBP22 and BiP, the major Hsp70 chaperone in the ER. The binding to BiP is mediated by the FKBP domain of FKBP22. Interestingly BiP enhances the chaperone activity of FKBP22. Both proteins form a stable complex with an unfolded substrate protein and thereby prevent its aggregation. These results suggest that BiP and FKBP22 form a folding helper complex with a high chaperoning capacity in the ER of Neurospora crassa.     

Mutations resistant to bromodeoxyuridine in mouse lymphoma cells selected by repeated exposure to EMS characteristics of phenotypic instability and reversion to HAT resistance by 5-azacytidine

Mutations induced by repeated EMS treatments were investigated by using mouse L5178Y cells. The frequency of TG^r mutations increased linearly with the number of EMS treatments whereas the yield of BrdUrd^r mutations showed a curvilinear dose-response curve. The BrdUrd^r frequency was roughly proportional to the square of the TG^r frequency and the results were compatible with the hypothesis that BrdUrd^r cells were induced by two mutational events within a cell. Most of the BrdUrd^r colonies isolated after 6 EMS treatments, however, were unstable. When BrdUrd^r colonies that had arisen in BrdUrd medium after 2 weeks' incubation were isolated in normal medium, the descendant cells showed a nearly normal level of thymidine incorporation and low plating efficiencies of about 1% in BrdUrd medium. In contrast, after isolation of the same colonies in BrdUrd medium, a low level of thymidine incorporation and high plating efficiencies in BrdUrd medium were observed in the descendant cells.

Reverse selection from BrdUrd^r to HAT^r was accomplished with frequencies of 10⁻⁶−10⁻³ for the descendants grown in BrdUrd medium, and AzaCyd treatment drastically increased the reversion frequency to nearly 10⁻¹. Further re-revertants from HAT^r to BrdUrd^r were also found with frequencies of 10⁻³−10⁻² without treatment. These results indicate that the initial BrdUrd^r cells did not result from inactivation of the thymidine-kinase gene but that the mode of gene expression was altered in some way.